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EARLY WARNING BIOMARKERS OF POLLUTION IN BIOMONITORING
PROGRAMMES. THEIR ECOTOXICOLOGICAL SIGNIFICANCE.
Ibon Cancio
Biologia Zelularra Atala, Zoologia eta Animali Zelulen Dinamika Saila, Zientzi
Fakultatea, Euskal Herriko Unibertsitatea, 644 P.K. E-48080 Bilbo (Basque Country).
zoxcauri@lg.ehu.es
In recent years, public interest in environmental pollution has grown very rapidly,
an interest that has been promoted by extensive coverage in the media. In this scenario,
a new science has been developed during the last 20 years, ECOTOXICOLOGY. At the
beginning ecotoxicology was only considered as a set of procedures that intended to
protect the environment through management and monitoring of pollutant discharges
into the environment.
Most recently, stimulated by the appearance of a public environmentalistic concern
politicians have rushed to create a legislative and administrative body with the intention
of protecting the environment from an increasing “polluting” human development. But
the industry is normally some strides ahead of this legislative will, and every single year
assists to the appearance of new polluting chemicals in the environment. Facing this
situation, the environmental manager needs rapid answers concerning the potential
toxicity and likely environmental impacts of a very broad range of chemicals and how
to control discharges.
A particular difficult problem is establishing the effects of chemicals in a complex
and variable environment, either upon individual organisms, or at population and
community levels. The use of biomarkers is an exciting development in ecotoxicology,
which can solve some problems. We could define biomarkers as sensitive and specific
tools that can be employed to measure exposure to pollutants –and sometimes can
measure toxic effects– using samples obtained from the field.
The ability to measure let us say, a characteristic biochemical response of a
determinate organism to a given change in its surrounding environment can provide the
evidence necessary to establish the presence or absence of such a change. This kind of
approach is in great progress through the utilization of the new techniques of molecular
biology, cell biology and biochemistry applied to measure the effects of chemicals on
human beings.
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SENTINEL SPECIES
The presence of toxic compounds in the environment can be measured by direct
chemical analysis of water and sediments, but this approach gives little information
about the bioavailability of these toxic chemicals. This means that it gives little
information about the concentration of pollutants susceptible of causing a damage in the
biota. Is in this point where sentinel species, or bioindicators, play an central role. We
consider a sentinel species:
.- A species that is able to bioaccumulate high concentrations of pollutants in its
tissues. That means that the bioindicator must be highly resistant to the polluting insult.
.- It occupies an important position in a given ecosystem and thus, is going to be
able to integrate the effects of a contaminant on that ecosystem.
Marine mussels are widely used in the so-called “Mussel-Watch” biomonitoring
programmes (Goldberg in 1975 proposed the concept of “Mussel Watch” to name the
global programme to monitor the water quality in coastal and estuarine environments
assessed by mussels) due to their ability to accumulate pollutants. This program has
been developed in nearly 100 sampling stations along the coasts of the United States
since then. Chemical analyses in biota is easier than chemical analyses of water and/or
sediments due to the bioaccumulating capacity of mussels. In general terms,
programmes based on the recording of contaminant levels in biological indicators have
been successfully tested at national and regional levels and appear at present as the only
type of monitoring which may be applied meaningfully on a global scale. The most
studied habitats have been near shore or estuarine habitats.
In the last years, bivalves in general (oysters, clams and most preferentially
mussels) have been found to be useful as indicators and integrators of exposure to
certain contaminants because:
1.- their wide geographic distribution,
2.- their dominant presence in coastal and estuarine communities,
3.- the fact of being filter-feeder able to accumulate pollutants in their tissues,
4.- their ability to respond against environmental pollution without showing
prolonged stress due to handling,
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5.- their importance as a economic food source for human beings,
6.- for being a sessile species,
7.- their occurrence in large populations able to provide enough data for short- or
long-term experiments, and
8.- they can be readily transplanted and maintained in cages in sites of interest.
Fish have also become an important target of study. Their economical value is out
of any doubt but they present a problem, they are mobile species and thus are not
supposed to be able to integrate the situation (in terms of cleanliness) within a given
location. It is assumed that fish under stress move away to places where living
conditions are better. The ability to move of fish is a problem that can be circumbented
employing cages for the maintenance of animals in locations of interest. Anyhow, any
strategy trying to assess the quality of a given environment should target on a series of
different biomarkers studied on different sentinel species placed at different levels in
their ecosystem. We propose to employ mussels, fish and polychaetes in marine and
estuarine ecosystems and fish, oligochaetes and crayfish in rivers.
Besides the chemical analysis of the pollutant burden in the biota we can employ
biomarkers. The use of biomarkers is a strategy that traks alterations in the animal
metabolism produced by the accumulation of pollutants in the cells. The interest of such
approach is that cells are placed in the interface between molecular events and events at
the organism level. Consequently, changes in cells anticipate and reflect what is going
to happen at higher levels of biological organization. According to this, in the last years
the aim of most biomonitoring programmes has been to develop new technologies to be
applied in order to assess environmantal pollution. The use of biomarkers is based on
the following principle: the effects produced by pollutants at the ecosystem level can be
promptly observed at lower levels of biological organization, that is at cell and tissue
levels. These effects are considered as early warning biomarkers of exposure to
pollutants (McCarthy and Shugart, 1990). In this context, approaches based on the
analyses of cells and/or tissues, such as the measurement of metallothionein induction,
structural alterations in the digestive lysosomal system, induction of cytochrome P450
system, the MFO system or antioxidant enzymes, genome alterations, peroxisome
proliferation, reduction in the thickness of digestive epithelia, the extent of metal
accumulation in digestive cell or hepatic lysosomes, or the induction of imposex
(hormonal changes caused by environmental pollutants that induce apparition of a non-
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functional penis in females leading to the dissapearance of the population), have been
applied successfully in some biomonitoring programmes conducted to detect metallic
and organic pollution. Under this framework fish have aquired special relevance. Fish
are very useful because they have livers. The liver is an organ profusedly studied in
mammalian organisms, and thus the biomarker approach can benefit from years of
research and knowledge on the cell and molecular biology of hepatocytes. In this
scenario, people centering their efforts in the digestive gland on mussels have been
struggling to keep pace with the wealth of data published on fish species.
We distinguish two different kinds of biomarkers: biomarkers of exposure and
effect biomarkes. Exposure biomarkers: are changes at cellular or molecular level that
indicate that the animal has been under exposure to contaminants. Effect biomarkers:
are changes at the cellular or molecular level that indicate that under a situation of
exposure to a contaminant the animal has been able to respond in a determinated way.
These biomarkers can be at the same way clasified as specific or as general biomarkers.
Specific biomarkers would be those indicating the precise cause of an observed
response (for instance there are biomarkers that indicate that an animal has been
exposed to heavy metals, or to organic pollutants or even to a specific compound like
for instance tributyltin or a carbamate insecticide). General biomarkers do not give
information about the exact cause of a response and only indicate that the animal has
undergone a stress situation.
The important feature of these biomarkers is that they can be employed in a
predictive way, allowing the initiation of bioremediation startegies before irreversible
environmental damage of ecological consequences occurs.
We shall pay attention now to a series of biomarkers, some of which are under
study in our laboratory in the University of the Basque Country and that we consider of
special interest. Recently, the biomarker approach has been incorporated into several
pollution monitoring programmes in Europe and the USA (North Sea Task Force
Monitoring Master Plan and the NOAA’s National Status and Trends Program) and
some of the biomarkers that are going to be described are being implemented
(Cajaraville et al., 2000). We shall focus in biomarkers of general stress, biomarkers of
exposure to heavy metals and biomarkers of exposure to organic pollutants (some of
these will be effect biomarkers).
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GENERAL STRESS BIOMARKERS
Biomarkers of stress should be of common use in pollution monitoring programmes
since they are able to define the health status of the animals on which we could then
apply exposure and effect biomarkers. We shall review lysosomal alterations as a
general biomarker used world-wide nowadays and cell type replacement a novel index
developed in our laboratory.
Lysosomal alterations: Lysosomes are ubiquitous cell organelles discovered by
Christian de Duve in the 60s. In aquatic organisms this organelle is predominant within
the digestive tissues of invertebrates (a very well developed lysosomal system is
localized within the digestive cells of molluscs) and the liver of fish. Their main
physiological function is the degradation of cellular macromolecules to low-molecular
weight products that can them be utilized in anabolic processes. This is the reason why
lysosomes are filled with hydrolitic enzymes (acid hydrolases) for the degradation of
carbohydrates, lipids and proteins. Lysosomes are also the main site of accumulation of
pollutants. The accumulation of pollutants may induce lysosomal enlargement and
lysosomal membrane destabilization, processes that have long been employed as
biomarkers of exposure to contaminants. This biomarker is considered as a general
biomarker of stress since lysosomal alterarions can be triggered in response to a variety
of stressors including organic and metallic pollutants, changes in the reproductive cycle
and changes in several natural environmental variables such as temperature, salinity or
food availability.
The technical approach to the quantification of these alterations include the
histochemical staining of a lysosomal activity (-glucuronidasem acid phosphatase,
hexosaminidase etc…) and the posterior quantification through automated image
analysis of the changes in the lysosomal compartment. This has been accepted as an
accurate and rapid technique for the measurement of pollutant impact on aquatic
organisms by the WHO-FAO and have been recommended for biological effects
monitoring in the Joint Assessment and Monitoring Programme (JAMP) developed by
the Oslo and Paris Comissions (OSPAR) (Stagg, 1998).
Cell type replacement. The digestive gland of molluscs is constituted by two or
three cell types depending on the species. In any case, one of the cells is mainly
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involved in intracellular digestion (digestive cell) while a different typo of cells is
mainly a secreting cell (secretory or basophilic cell). The digestive gland is the main
target organ of pollutants accumulating in molluscs. These pollutants tend to be
accumulated within the profusedly developed lysosomal compartment localized in the
digestive cells, where in cases of acute exposure they could trigger toxic responses. We
have been able to detect, in animals exposed to different kinds of chemicals (either
organic or heavy metals) and in animals inhabiting polluted sites, that the animal suffers
changes in the ratio digestive to secretory cells in their digestive glands. Digestive cells
are normally more abundant than secretory ones, and for instance slugs inhabiting for
generations in an abandoned copper mine in Wales only present secretory cells
(Marigómez et al 1998). Similarly, mussels exposed in the lab to Cd or to
benzo(a)pyrene substitute the existing digestive cells for basophilic cells within the few
days of exposure, in a mechanism that ensures that the contaminants will not be
accumulated within the tissues. This is a mechanism of general stress that is necessary
to have in mind, since low concentrations of pollutants within tissues do not always
reflect a clean environment, but the onset of a survival adaptation mechanism to
preclude the toxic effects of the pollutants.
BIOMARKERS OF EXPOSURE TO ORGANIC POLLUTANTS
Organic pollutants accumulate readily into biological structures. Due to their
hydrophobicity they tend to bind to lipophilic structures, this meaning that they bind
strongly to tissues with high fat content such as gonads, digestive tissues or liver. At the
subcellular level, cell membranes, lipid inclusions and lysosomes are the main
accumulation compartments.
The range of organic products to be found in the environment is impresive and
include
pesticides,
fungicides,
insecticides,
herbicides,
polycyclic
aromatic
hydrocarbons (PAHs), polychlorinated biphenils (PCBs), bleached kraft mill effluents,
seawage effluents, plastisizers, solvents, food flavours, drugs of different classes and
structures… Whatever you think about, you have it. The chemical analysis for the
identification of this millieu of compounds is extremely expensive, very difficult and is
bound to be aimed to localise a compound that is not in our environment while instead
we have thousands of others. This is the reason that makes the biomarker approach
extremely important specially in the localization of exposure to organic compounds. We
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shall review in this text acetylcholinesterase inhibition, cytochrome P450 system
induction, imposex, peroxisome proliferation and vitellogenin expression in male fish.
Acetylcholinesterase (AChE) inhibition: AChE catalizes the hydrolisis of
acetylcholine into choline and acetic acid. Its inhibition is directly linked with the
mechanisms of toxic action of organophosphorus and carbamate insecticides. Several
studies hace succesfully used AChE inhibition as a tool to diagnose organophosphorus
pesticide poisoning in birds and fish.
Cytochrome P450 system induction: The cytochrome P450 monooxygenase
system is constituted by a family of structurally and functionally related heme proteins.
Multiple protein subfamilies within this system participate in the oxidative metabolism
of diverse substrates, including drugs, xenobiotics and physiological substrates such as
steroids, neurohormones, fatty acids and prostaglandins. The cytochrome P4501A
(CYP1A) subfamily plays a central role in the biotransformation of many organic
xenocompounds including dioxins, furans, polychlorinated biphenyls and polycyclic
aromatic hydrocarbons. Under exposure to these compounds the CYP1A system is
transcriptionaly activated via a nuclear hormone receptor, termed arylhydrocarbon
receptor (Ah). The induced CYP1A protein converts lipophilic organic xenobiotics in
more water soluble compounds that can be readily excreted and detoxified through the
urine. However, some of the intermediate products of these metabolic pathways can be
more toxic than the parent compound.
Induction of CYP1A has been observed in fish liver exposed to certain classes of
organic contaminants. In this way, EROD activities or CYP1A protein levels in fish
collected in field studies have been correlated with environmental levels of CYP1Ainducing chemicals such as PAHs or PCBs. Nowadays, this biomarker is well-known
and of widespread use although there is a need to be carefull aplying this biomarker
since factors such as temeperature, season or sexual hormones can also modulate the
fish CYP1A system responsiveness.
The main problem of this biomarker is that the cytochrome P450 system, and
specially the CYP1A system, have no special relevance in molluscan tissues, where no
consistent data have been published. Thus, the most important sentinel species, the
mussel, does not present an important biotransfromation system in its endoplasmic
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reticulum, discouraging the utilization of this biomarker in pollution biomonitoring
programmes.
Imposex. Imposex is one of the most specific biomarkers to date. Whenever we
localize imposex, we can say that it is due to exposure to organotin compounds, most
probably to trubutyltin (TBT). Imposex refers to the imposition of male characteristics
on female gastropod molluscs and it has been identified in more than 150 species of
marine prosobranch snails world-wide (Matthiessen et al. 1999). These species show
different sensitibities to TBT. Imposex results normally in the growth of a penis in
female organisms that in some species results in the appearance of a superficial vas
deferens that can obliterate the oviduct. In such cases reproduction is impossible and the
viability of the population is in jeopardy.
TBT is one of the most potent androgenic compounds ever added to the
environment. It was widely used as active ingredient in the antifouling paint applied
bellow the waterline on ship hulls. The use of TBT in ships smaller than 25 meters was
long ago restricted and for instance the UK banned its use for small vessels in 1987.
TBT is considered to induce imposex through the inhibition of the enzyme aromatase,
inhibiting in this way the metabolism of testosterone to 17-estradiol. This would lead
to increase testosterone concentrations and masculinization.
Induction of vitellogenesis in male fish. In the last years a series of chemical
products have hit the news due to their health risk effects, they are termed endocrine
disrupters
(environmental
oestrogens;
endocrine
modulators,
ecoestrogens,
environmental hormones, xenoestrogens, hormone-related toxicants or phytoestrogens).
An endocrine disrupter is a substance, or a mixture of them, that alters function of the
endocrine system and consequently causes adverse health effects in an intact organism,
or its progeny, or (sub)populations. Among the most potent endocrine disrupters we
find some natural products such as coumestrol and genistein, pharmaceuticals such as
diethylstilbestrol, 17-ethynylestradiol and tamoxifen, and industrial chemicals such as
DDT, bisphenol A, methoxychlor, chlordecone, alkylphenols, PCBs and dibutyl
phthalate (Vos et al 2000).
The interest in these compounds was fired when some fish species within some
British rivers shown an alarming case of sex determination. All the individuals showed
female features. Something similar, accompanied by a tremendous decline in juveniles,
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occurred with the population of alligators inhabitting lake Apopka in Florida. In the first
case, seawage effluents were blamed (most specifically alkylphenols and 17ethinylestradiol -synthetic hormone deriving from contraceptive agents); in the second
case DDT coming from agricultural activities was taken as responsible for these
alterations. The cases could be considered as anecdotical (although similar responses
have been observed world-wide mainly in fish, reptilian and bird species) if it was not
for statistical studies that have shaken our anthropogenic understanding of the
environment. The case is that although the last years have experimented a decrease in
the amount of cancers in the human population, a close look at the cases reveal that the
amount of cancers related to sexual organs have increased (prostate, ovarian, breast…).
Interestingly, people inhabiting areas of high pesticide input show higher chances of
suffereing any of these cancers. Yet another observation has triggered politicians in
USA and Europe to a race to regulate endocrine disrupters. We can not forget that most
politicians are male and the last decade has witnessed a decline of the sperm production
in the human population. Although we do not know the effect that such decline could
have on human population viability, it is out of any doubt that it has been an observation
that has flashed all the red lights.
It is difficult to trace the cause of a cancer since the etiology of the disease is very
complicated. To stablish a link between endocrine disrupters and cancer is as difficult as
it is to stablish it between cigarretes and cancer (depending on the attorney or press
responsible that defends the polluting company).
On the other hand, a possible link between a potential endocrine disrupter and
changes in the endocrine system of a fish species is easier to stablish. Endocrine
disrupters can mimic the natural hormones by binding to hormone receptors or
influencing cell signalling pathways. They can block, prevent and alter hormonal
binding to hormone receptors or influence cell signalling pathways. Moreover, they
could alter the production and breakdown of natural hormones (as it is the case of TBT
in gastropods) or alternatively they could modify the production and function of
hormone receptors.
In the case of fish, as it is the case also in some reptiles and in invertebrates, sex
determination and differentiation are less geneticaly characterized and are influenced by
environmental factors such as temperature, food availability and, of course, by the
presece of xenobiotics with hormonal activity. The synthesis of the egg yolk precursor
protein vitellogenin, a exclusively female protein in fish, is considered a sensitive
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biomarker for estrogen exposure in male fish. Appearance of high vitellogenin levels in
male fish liver preceeds appearance of ovary tissue in those animals, and thus can be
employed as an early warning biomarker of exposure to environmental estrogens or
xenoestrogens.
Peroxisome proliferation: Peroxisomes are highly heterogeneous organelles that
vary in size, shape and protein composition depending on species, tissue, cell, metabolic
state or developmental stage (Cancio and Cajaraville 2000). In general, peroxisomes are
single membrane-bounded organelles with a finely granular electron-dense matrix. De
Duve and Baudhuin (1966) defined peroxisomes as organelles containing at least one
flavin oxidase that produces H2O2 and catalase that degrades it. Nowadays, the term
microbody is applied to all vesicles with common characteristics of peroxisomes,
microperoxisomes, glyoxysomes of plant seeds and glycosomes from trypanosomatids.
Liver parenchymal cells have been extensively studied as the cell type showing the
highest accumulation of peroxisomes, which represent 1.5% of the parenchymal cell
volume and 2.5% of the total liver protein.
Peroxisomes are plastic organelles that may undergo extensive proliferation
(increase in number), accompanied by induction of some enzyme activities (most
dramatically the 3 enzymes of the peroxisomal -oxidation). Peroxisome proliferation
was first documented in the liver of rats exposed to a hypolipidemic drug, clofibrate.
Posteriorly it attracted increasing interest when a link between the proliferation process
and carcinogenesis was established in rodents. The array of chemicals that induce
proliferation is impressive and no evident structural similarities can be observed among
them with the exception of an acidic group, generally a carboxyl group. The compounds
traditionally employed as peroxisome proliferators receive the generic name of
hypolipidemic drugs and are therapeutically used in cases of arteriosclerosis and
hipercholesterolemia. A different group of compounds extensively studied for their
proliferating activity are plasticizers among which phthalate esters are the most
important. Other classes of peroxisome proliferators are steroids, pesticides, solvents
and diverse industrial chemicals, food flavors, several therapeutic drugs, hydrocarbons
and natural products. High fat diets, cold adaptation, vitamin E deficiency, riboflavin
deficiency, genetic obesity, diabetes and starvation can also lead to peroxisome
proliferation in rodents.
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Peroxisome proliferation in mammalian cells is associated with changes in lipid
homeostasis as a result of the activity changes in enzymes handling fatty acids, mainly
the induction of peroxisomal -oxidation and microsomal -oxidation. All three
enzymes of the peroxisomal -oxidation are induced, both at the protein and at the
mRNA level, reaching 30-fold activity increases. All this alterations are mediated
through a nuclear hormone receptor, the peroxisome proliferator activated receptor
(PPAR).
Peroxisome proliferation has been observed in the liver of fish and the digestive
gland of molluscs exposed to organic pollutants. Some of the chemicals described to
induce peroxisomal response in aquatic animals under laboratory conditions are
pesticides and insecticides such as dieldrin, paraquat, endosulfan or carbamate; and
PAH-s such as benzo(a)pyrene, crude oils or commercial lubricant oils. In the same
way, increased peroxisomal parameters have been correlated to high concentrations of
PAHs and PCBs in field studies.
We consider peroxisome proliferation as a useful, early warning biomarker of
exposure to organic pollutants that can be used instead of the now more successful
cytochrome P450 system induction both in fish and in mollusc tissues.
BIOMARKERS OF EXPOSURE TO HEAVY METALS
The body content of a trace metal in a given organism results from the net balance
between the processes of metal uptake and metal loss. Consequently, the amount of
metals accumulated by aquatic organisms also depends upon abiotic factors (metal
solubility, metal speciation, complexation...) and biotic factors (growth, biochemical
composition, reproductive condition, metabolism, excretion...). Understanding the
strategies adopted by cells in their involvement with metals, is the key to assess the
ecotoxicological significance of accumulated metal burdens commonly reported in
biomonitoring programmes.
It seems that most of the metal containing particulate material is taken up via
endocytosis, an active transport mechanism that requires energy. Transfer of the metals
across the plasma membrane of the cell probably occurs via facilitated diffusion.
Accordingly, the plasma membrane contains intrinsic proteins through which ions
selectively pass. Therefore, certain metals can cross the membrane by means of
membrane-transport proteins. Particulate metals (i.e. Cd) may employ ion pumps or the
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endocytic processes to cross the cell membrane. Dissolved metals can be uptaken by
diffusion, carrier-mediated active transport mechanisms or pinocytosis. Once within the
cell, soluble metals bind to a variety of inorganic ligands or to low molecular weight
organic compounds. Alternatively, this metals can be neutralized in specific cell types
or be engulfed within calcium concretions. The rates of metal accumulation are
determined by the number and binding characteristics of the available ligands and their
accessibility to the metals. In some cases, metal uptake may be enhanced by the
synthesis of low molecular weight binding proteins known as metallothioneins. In the
case of the endocytic pathway, the endocytic vesicles fuse with primary lysosomes to
give heterophagolysosomes in which the biological material is degraded. In this way,
the metal is partially available for the cell and partially bound to the undegraded
material that remains in the lysosomal vacuolar system, which is finally eliminated from
the cell via exocytosis of residual bodies. Thus, the cellular distribution of the metal
may vary greatly depending on the metal retention characteristics of the lysosomes.
The determination of the specific cell or tissue compartments involved in metal
metabolism could be an accurate tool to assess the metal bioavailability in the
environment by analyzing/quantifying metal levels found in those selected target
compartments. We shall focus here on the autometallographical quantification of black
silver deposits and on metallothionein induction.
Autometallographical
quantification
of
black
silver
deposits
Some
histochemical analyses allow an accurate determination of the metal levels in biological
samples. Autometallography is a method based on the self-induced silver amplification
of protein-bound metal ions (except Ca) in biological sections that are visualized as
black silver deposits (BSD). The extent of BSD in digestive cell lysosomes of molluscs
or crustaceans and in hepatic lysosomes of fish quantified by automated image analyses
could be used as an index for routine screening of metal pollution, and to accurately
reflect the total concentration of metals in the digestive gland of mussels,
hepatopancreas of crustaceans or liver of fish, and concomitantly, the fraction of
bioavailable metals for the biota in sea or freshwater. These methods provide quick and
cost-effective alternatives to routine chemical analyses. Applying autometallographic
techniques environmentally relevant concentrations of heavy metals have been
visualized within animal tissues, and differences among animals coming from
environments with different heavy metal burden have been easily detected. We propose
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that this cost-effective biomarker could be employed in pollution biomonitoring
programmes as indicative of possible heavy metal pollution. In case of negative results
no further measures should be taken, but in the case that increases in the extent of BSD
were detected we could employ chemical techniques (more expensive, more technical,
skill dependant and cumbersome) for the determination of the heavy metal species that
in each case were responsible of the observed response.
Metallothionein induction. Metallothionein (MT) is a low-molecular weight
protein very rich in cysteine groups (>20-30%) that possesses a selective binding
affinity for zinc, copper and other group II heavy metal ions. MTs are considered central
in the intracellular regulation of metals such as copper, zinc, and cadmium. Increased
MT synthesis is associated with increased capacity for binding these metals and
protection against metal toxicity. MTs are supposed to bind metals in the cytosol which
then are transported to the lysosomal compartment. Another functions are the role in the
immunitary system and the neutralization of free radicals and the cellular storage for
essential metals. MTs have been detected in approximately 50 different species of
aquatic invertebrates, the majority of which are molluscs or crustaceans. Of course,
MTs have also been detected in fish species. Strong inductions in the synthesis of MTs
have been observed in a great number of molluscs and fishes exposed either in vivo or
under laboratory conditions to heavy metals. Anyhow, the degree of MT induction can
vary between species and between tissues. The involvement of MT in metal
sequestration is more evident in the gills, digestive gland and kidney, reflecting the
significance of these tissues in uptake, storage and excretion of metals. The
quantification of MTs in fishes and molluscs has been applied as a reliable index of
metal exposure to be applied in biomonitoring programmes (JAMP, MEDPOL). MT
induction in animal tissues can be quantified by different analytical methods. By
differential pulse polarography the –SH groups within the MT can be quantified
presenting an estimate of the protein concentration in the tissue. With an specific
antibody against the protein in a given species radioimmunoassay, ELISA or Western
blot approaches can be employed.
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FUTURE PERSPECTIVES IN BIOMONITORING PROGRAMMES
One of the largest scale biomonitoring programmes currently under application, the
MED POL, considers a number of biomarkers starting from those capable of giving a
general indication of biological stress due to pollution. These general stress indices
include the assessment of damage to genetic and subcellular components. On the other
hand, the elevation of enzymatic activity of the cytochrome P450 system (organic
pollution) and the induction of metallothionein protein synthesis (heavy metals
pollution) are considered as specific stress indices. These biomarkers of pollution seem
to offer the best information on adverse changes to normal genetic, biochemical and
cellular systems in marine organisms as early warning signals that environmental
damage is in progress.
The next years will assist to a change in the monitoring programmes that will
incorporate these effect biomarkers to the currently employed exposure biomarkers
(pollutant burden in bioindicator species). There is a need to plan and develop a
multidisciplinar monitoring programme to assess environmental pollution. First, we
should agree on the selection of the most adequate sentinel species. As a rule for
estuarine environments it is clear that the target species should be the blue mussel
(Mytilus edulis or galloprovinicialis). In cases where there is no presence of the blue
mussel other bivalve species could be employed, such as Ruditapes philippinarum or
Ruditapes decussatus. In addition, a gastropod mollusc should be employed to
investigate possible presence of TBT in estuarine environments.
As molluscs show a limited capacity to metabolize organic xenobiotics when
applying the induction of the cytochrome P450 system it seems sensible to employ a
fish species and in such cases the dab Limanda limanda or the red mullet Mollus
barbatus have been employed in marine environments. In rivers the stickelback, the
rainbow trout (Onchorrynchus mykiss) or the carp (Ciprinus carpio) could be the
species of interest. These studies should be complemented with the study of polychaetes
in estuarine environments and oligochaetes and crayfishes in rivers.
The next step would be the selection of significant biomarkers, some of which have
been reviewed here. Some of these biomarkers are used world-wide and have already
been implemented in some biomonitoring studies, others need further developments
before implementation. For quality assurance there would be a need to intercalibrate the
different biomarkers among different laboratories and in different ecosystems. In
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general, it seems that no individual biomarker can give a complete diagnosis of
pollution effects in running waters. Rather, a set of biomarkers should be employed
together with other chemical and biological measurements.
Finally, it is fundamental to develop mathematical instruments to objectively and
correctly evaluate the effects of pollution on the health or aquatic organisms by
integrating the results obtained with different biomarkers. We must also necessarily
have a detailed knowledge of basal values of the biomarkers and consider the seasonal
variations of biomarkers in order to distinguish pollution-related effects from natural
fluctuations. This could be achieved by developing an adequate database for defining
normal ranges of variation of selected biomarkers in field conditions.
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